U.S. patent application number 11/016781 was filed with the patent office on 2006-06-22 for extraction, separation and modification of sweet glycosides from the stevia rebaudiana plant.
This patent application is currently assigned to Stevian Biotechnology Corporation Sdn. Bhd. Malaysia. Invention is credited to Lidia A. Abelyan, Varuzhan H. Abelyan, Mariam O. Adamyan, Vahe T. Ghochikyan, Avetik A. Markosyan.
Application Number | 20060134292 11/016781 |
Document ID | / |
Family ID | 36596166 |
Filed Date | 2006-06-22 |
United States Patent
Application |
20060134292 |
Kind Code |
A1 |
Abelyan; Varuzhan H. ; et
al. |
June 22, 2006 |
Extraction, separation and modification of sweet glycosides from
the stevia rebaudiana plant
Abstract
The invention disclosed relates to a method for the extraction
of sweet glycosides from the Stevia rebaudiana Bertoni plant and
recovery of individual rebaudioside A and stevioside. The
extraction is developed in the presence of pectinase, and the
extract is purified using cyclodextrin and bentonite. High purity
rebaudioside A is obtained by crystallization and recrystallization
from ethanol. High purity stevioside is prepared from the filtrate
by purification with cyclodextrin, bentonit, and ion exchange
resins. The enzymatic modification of the rebaudioside A,
stevioside and the purified extract is carried out using the
transferring enzymes derived from Thermoactinomyces vilgaris and
Bacillus halophilus.
Inventors: |
Abelyan; Varuzhan H.;
(Negeri Sembilan, MY) ; Ghochikyan; Vahe T.;
(Negeri Sembilan, MY) ; Markosyan; Avetik A.;
(Negeri Sembilan, MY) ; Adamyan; Mariam O.;
(Negeri Sembilan, MY) ; Abelyan; Lidia A.; (Negeri
Sembilan, MY) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Stevian Biotechnology Corporation
Sdn. Bhd. Malaysia
Negeri Sembilan
MY
|
Family ID: |
36596166 |
Appl. No.: |
11/016781 |
Filed: |
December 21, 2004 |
Current U.S.
Class: |
426/548 |
Current CPC
Class: |
A23L 27/36 20160801;
A61K 36/28 20130101; A23L 33/105 20160801 |
Class at
Publication: |
426/548 |
International
Class: |
A23L 1/236 20060101
A23L001/236 |
Claims
1. A process for recovering sweet glycosides from Stevia rebaudiana
plant material, which comprises the following steps: a) treating
the Stevia rebaudiana plant with hot water to extract an aqueous
liquid solution containing mixed sweet glycosides; b) enzymatic
treatment of the extract with pectinase during the extraction; c)
contacting the extract from step (b) with beta cyclodextrin; d)
treating the aqueous extract with bentonite; e) evaporating the
filtrate from step (d) to syrup state or dryness.
2. A process for the purification of rebaudioside A and stevioside,
which comprises the following steps: a) dissolving the mixture of
sweet glycosides obtained in step (1e) with ethanol, heating the
solution and then cooling the solution to precipitate Rebaudioside
A; b) filtering the solution to recover the precipitate
Rebaudioside A and a filtrate containing other sweet glycosides; c)
suspending the crystallin Rebaudioside A obtained in step (2b) in
ethanol-water solution, heating and then cooling the solution to
precipitate the Rebaudioside A at a higher purity; d) recovering
the higher purity Rebaudioside A. e) treating the solution obtained
in step (b) with bentonite; f) treating the solution obtained in
step (b) with beta cyclodextrin; g) desalting and concentrating the
filtrate from step (f) by heating and evaporating to dryness to
obtain powdered Stevioside; h) suspending the crystallin stevioside
obtained in step (2) in ethanol, heating and then cooling the
solution to precipitate the Stevioside at a higher purity; i)
recovering the higher purity Stevioside.
3. A method for the transglucosylation of Stevia sweet glycosydes
including stevioside or rebaudioside A or purified extract
treatment with transferring enzyme from the microorganisms
Thermoactinomyces vulgaris INMIA-Tac-3554 in the presence of
carbohydrates, purification and drying of the final products;
4. A method of claim 3 wherein the transferring enzyme produced by
Bacillus halophilus BIO-12H is used as biocatalyst.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a method for the extraction of
sweet glycosides from the Stevia rebaudiana Bertoni plant and
recovery of individual rebaudioside A and stevioside.
[0002] The present invention also relates to a novel transferring
enzymes capable of catalyzing the efficient transglucosylation of
stevioside, rebaudioside A and the mixture of glycosides.
BACKGROUND OF THE INVENTION
[0003] The worldwide demand for high potency sweeteners is
increasing and, with blending of different sweeteners becoming a
standard practice, the demand for alternatives is expected to
increase. The sweet herb of Paraguay, Stevia rebaudiana Bertoni,
produces an alternative sweetener with the added advantage that
Stevia sweeteners are natural plant products. In addition, the
sweet steviol glycosides have functional and sensory properties
superior to those of many high potency sweeteners.
[0004] The sweet diterpene glycosides of Stevia have been
characterized and eight sweet glycosides of steviol have been
identified. These glycosides accumulate in Stevia leaves where they
may comprise from 10 to 20% of the leaves dry weight. On a dry
weight basis, a typical profile for the four major glycosides found
in the leaves of Stevia comprises 0.3% dulcoside, 0.6% rebaudioside
C, 3.8% rebaudioside A and 9.1% stevioside. Other glycosides
identified within Stevia include rebaudioside B, C, and E, and
dulcosides A and B. Rebaudioside B may be an artifact formed from
rebaudioside A during extraction since both rebaudioside A and
rebaudioside D are found to convert to rebaudioside B by alkaline
hydrolysis.
[0005] Of the four major diterpene glycoside sweeteners present in
Stevia leaves only two, stevioside and rebaudioside A, have had
their physical and sensory properties well characterized.
Stevioside and rebaudioside A were tested for stability in
carbonated beverages and found to be both heat and pH stable (Chang
and Cook, 1983). Stevioside is between 110 and 270 times sweeter
than sucrose, rebaudioside A between 150 and 320 times sweeter than
sucrose, and rebaudioside C between 40 and 60 times sweeter than
sucrose. Dulcoside A was 30 times sweeter than sucrose.
Rebaudioside A was the least astringent, the least bitter, had the
least persistent aftertaste and was judged to have the most
favorable sensory attributes of the four major steviol glycosides
(Phillips, 1989 and Tanaka, 1997). Dubois and Stephanson (1984)
have also confirmed that rebaudioside A is less bitter than
stevioside and demonstrated that the bitter notes in stevioside and
rebaudioside A are an inherent property of the compounds and not
necessarily the result of impurities in whole plant extracts.
Bitterness tends to increase with concentration for both stevioside
and rebaudioside A. Both stevioside and rebaudioside A are
synergistic in mixtures with other high potency sweeteners such as
aspartame and are good candidates for inclusion in blends
(Schiffman et al. 1995).
[0006] A process for the recovery of diterpene glycosides,
including stevioside from the Stevia rebaudiana plant is described
(U.S. Pat. No. 4,361,697). A variety of solvents, having different
polarities, were used in a sequential treatment that concluded with
a high performance liquid chromatographic (HPLC) separation
procedure.
[0007] The method for the recovery of rebaudside A from the leaves
of Stevia rebaudiana plants is developed (U.S. Pat. No. 4,082,858).
Again, final purification is achieved by liquid chromatography
subsequent followed by an initial extraction with water an alkanol
having from 1 to 3 carbon carbons, preferably methanol. It is also
disclosed that water may be used as the initial solvent, their
preferred solvent at this stage is a liquid haloalkane having from
1 to 4 carbon atoms. The preferred second solvent is an alkanol
having from 1 to 3 carbon atoms, while the preferred third solvent
is an alkanol having from 1 to 4 carbon atoms and optionally minor
amounts of water.
[0008] U.S. Pat. No. 4,892,938, to Giovanetto discloses a
purification process in which the aqueous extracts of the plant are
purified by passing these aqueous extracts through a series of
ion-exchange resins which are selected to remove various
impurities. The sweet glycosides remain in the water and are
recovered by evaporation of the water. The advantage is that
everything is done in water, while most other processes involve the
use of a solvent at some point. The disadvantage is that the final
product is quite impure with only about 70% is a mixture of the
sweet glycosides. The balance is mainly material more polar than
the sweet glycosides which we have identified as a complex mixture
of polysaccharides (about 25%), and a small amount of yellow, oily
material less polar than the sweet glycosides (about 5%).
[0009] The low polarity oil was isolated by chromatography. The
flavor of the low polarity oil is very unpleasant. We have found
this oil to be present in varying levels from 0.2 to 2.0% in every
commercial product we have examined. Since of varying amounts this
intensely off-flavored material is contained in the commercial
materials it presents problems with quality control and flavor
issues. The polysaccharide fraction also appears to contain
off-flavor materials, but not as intense in flavor as the low
polarity yellow oil.
[0010] The sweet glycosides obtained from Giovanetto process are
always a mixture. We have determined that the two principle sweet
glycosides are Stevioside and Rebaudioside A, and two of the minor
sweet glycosides are Dulcoside and Rebaudioside C, although there
are many other minor ones. We have isolated the two principle
glycosides and we have found that there is a considerably different
flavor between them with one being much more desirable than the
other. Stevioside has an aftertaste which is undesirable. This
aftertaste is present in Stevioside samples of even greater than
99% purity. On the other hand, Rebaudioside A does not possess an
aftertaste and has a sweetness flavor comparable to sucrose. Thus
it is recognized as having the most desirable sensory properties.
In addition to this complexity, various impurities are also present
and some of these possess undesirable flavors. The entire matter is
further clouded by the extreme difficulty of doing analyzes. The
analytical exercise pushes at the envelope of present technology
and involves considerable art. Finally, the problem with the
methods described above is that the resulting materials contain a
mixture of all of the sweet glycosides.
[0011] The combined use of microfiltration, ultrafiltration, and
nanofiltration is also applied for the purification of stevia
extract (U.S. Pat. No. 5,972,120). The method gives a good result,
however the application of the above mentioned equipments makes the
product cost very expensive. Besides, the process again provided to
isolate only the mixture of glycosides, but not pure individual
compounds, such as stevioside and rebaudioside A.
[0012] Individual sweet glycosides are obtained from the stevia
rebaudiana plant. A mixture of sweet glycosides extracted from the
stevia rebaudiana plant is processed to remove impurities by using
two ion exchange columns. After removing the mixed sweet glycosides
from the second column with methanol the solution is dried. Upon
refluxing the dried solids in a methanol solution and then cooling
the solution, Stevioside precipitates out. The filtrate is further
concentrated and cooled to precipitate out Rebaudioside A. This
Rebaudioside A can be further purified as can the previously
obtained Stevioside (U.S. Pat. No. 5,962,678). However, a large
amount of toxic organic solvent, such as methanol is used.
[0013] However, stevioside possesses residual bitterness and
aftertaste, which affect its qualitative characteristics. They can
be eliminated by the reaction of intermolecular transglycosylation
of various enzymes, upon which the attachment of new carbohydrates
at positions C13 and C19 takes place. It is the number of
carbohydrate units in the above-mentioned positions that determines
the quality and degree of component's sweetness.
[0014] Pullulanase, isomaltase (Lobov et al., 1991),
.beta.-galactosidase (Kitahate et al., 1989), and dextrine
saccharase (Yamamoto et al., 1994) are used as transglycosylating
enzymes, with pullulan, maltose, lactose, and partially hydrolyzed
starch, respectively, being as donors.
[0015] The treatment with pullulanase results in production of
13-O-[.beta.-maltotriosyl-(1,2)-.beta.-D-glucosyl]-19-O-.beta.-D-glucosyl-
-steviol;
13-O-[.beta.-maltosyl-(1,2)-.beta.-D-glucosyl]-19-O-.beta.-D-glu-
cosyl-steviol and
13-O-[.beta.-sephorosyl-19-O-.beta.-maltotriosyl-steviol. Although
the yields of the transglycosylated products were rather low, the
selectivity in terms of the yield of the desirable mono- and
di-derivatives was higher than in the case of CGTase (Lobov et al.,
1991).
[0016] In case of maltase, three transglycosylated products are
also produced, namely
13-O-[.beta.-sephorosyl-19-O-.beta.-isomaltosyl-steviol;
13-O-[.beta.-isomaltosyl-(1,2)-.beta.-D-glucosyl]-19-O-.beta.-D-glucosyl--
steviol and
13-O-[.beta.-nigerosyl-(1,2)-.beta.-D-glucosyl]-19-O-.beta.-D-glucosyl-st-
eviol.
[0017] The transglucosylation of stevioside was also done by action
of cyclodextrin glucanotransferases (CGTase) produced by Bacillus
stearothermophilus FERM-P No 2222 (U.S. Pat. No. 4,219,571).
However, the commercialized stevioside consisting of roughly equal
amounts of purified stevioside and lactose was used as substrate,
and the transferring reaction is not investigated on pure
stevioside, pure rebaudioside A, and purified stevia mixture.
[0018] The object of the present invention is to provide an
advantageous process for the extraction of sweet glycosides from
Stevia rebaudiana Bertoni plant, and for the isolation of
stevioside and rebaudioside A, and a second object of the present
invention is to provide a novel transferring enzyme produced by
Themoactinomyces vulgaris and Bacillus halophilus which catalyzes
the transglucosylation of stevioside and rebaudioside A, as well as
the mixture of the glycosides obtained after extraction.
SUMMARY OF INVENTION
[0019] The present invention relates to a method of increasing the
rate of extraction for the recovering diterpene glycosides from
Stevia rebaudiana plant, separation of stevioside and rebaudioside
A, and their enzymatic modification. The invention seeks to
simplify the processes.
[0020] In accordance with the present invention, a process for
recovering sweet glycosides from the Stevia rebaudiana plant
material is provided. The dried and powdered leaves are treated
with water in the presence of a pectinase, cellulase, and
.alpha.-amylase. The use of such enzymes considerably increased the
extraction rate and facilitates the next stages of purification.
The resulted extract is purified using treatment with calcium
hydroxide and ultrafiltration. The permeate is passed through the
column packed with bentonite and concentrated to syrup state under
vacuum. The treatment with ethanol allows separating the
practically pure rebaudioside A from the mixture. The rebaudioside
A with high purity is obtained after washing the crystals with
88-95% of ethanol.
[0021] From the remaining solution stevioside is isolated after
evaporation of ethanol, deionization by ion exchange resins,
treatment with bentonite and spray drying. The high purity
stevioside is obtained after washing the crystals with 97-98% of
ethanol.
[0022] For the producing the mixture of sweet glycosides the
treatment with ion exchangers and bentonite is carried out without
isolation of rebaudioside A.
[0023] The application of cyclodextrin glucanotransferases of
Thermoactinomyces vulgaris and Bacillus halophilus with high
transferring activity allows increasing of the degree of
transglycosylation of stevioside and decreasing of the time of the
reaction.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows a high-performance liquid chromatographic chart
of purified extract;
[0025] FIG. 2 shows a high-performance liquid chromatographic chart
of rebaudioside A after the first crystallization;
[0026] FIG. 3 shows a high-performance liquid chromatographic chart
of rebaudioside A after recrystallization;
[0027] FIG. 4 shows a high-performance liquid chromatographic chart
of remaining solution after precipitation of rebaudioside A;
[0028] FIG. 5 shows a high-performance liquid chromatographic chart
of stevioside after the first crystallization;
[0029] FIG. 6 shows a high-performance liquid chromatographic chart
of stevioside after recrystallization.
[0030] FIG. 7 shows a high-performance liquid chromatographic chart
of a CGTase-treated stevioside;
[0031] FIG. 8 shows a high-performance liquid chromatographic chart
of a CGTase-treated rebaudioside A;
[0032] FIG. 9 shows a high-performance liquid chromatographic chart
of a CGTase-treated purified extract.
[0033] FIG. 10 shows the morphological characteristics of Bacillus
halophilus BIO-12H. (a) is vegetative cells; (b)--sporulated cells
and spores, .times.1000.
[0034] FIG. 11 shows the morphological characteristics of
Thermoactinomyces vulgaris INMIA-Tac-3554.
DETAILED DESCRIPTION OF THE INVENTION
[0035] Diterpene glycosides, including sweet-testing substances,
are found in the stems, seeds and leaves of the S. rebaudiana
plant, being present in the highest concentration in the leaves.
The leaves, therefore, are the preferred starting material for
recovery of sweet glycosides. It is preferred that plant material
be air dried before extraction, preferably at the temperatures
between 50-60.degree.C. for a period 2-3 hours to a moisture
content of 5-8%. Under these conditions the sweet glycosides are
not decomposed. It has been found that the smaller the size of
leaves the higher the rate of extraction. Mesh sizes of 20-30 (U.S.
Sieve Series) are preferred. The proportion of extraction water
preferably is about 5 liters to about 15 liters (pH 6.0 to 7.0) to
one kilogram of leaves. Higher volumes of solvent can be used
however it is not preferable from the practical standpoint. The
duration of extraction may be from 0.5 hours to 24 hours, with a
period of from about 1 hour to about 6 hours preferred.
[0036] The extraction temperature can be in the limits of
25-90.degree.C, however the temperatures between 45-75.degree.C are
more preferable.
[0037] The filtration rate and the clarification of extracts are
enhanced when extraction is done in the presence of commercial
pectinase (made by NOVOZYMES under the trade name of PECTINEX
Ultra-SP-L) in the amount between 1-4 grams per one liter,
preferably about 2 grams.
[0038] The plant material is separated from the solution by
filtration and the pH of the filtrate is adjusted to about 10 with
calcium hydroxide and heated between 40-60.degree.C, preferably
from 50.degree.C to 55.degree.C, for about 0.5-1.5 hours, cooled to
ambient temperature with slow agitation, and finally filtered.
[0039] The pH of resulted filtrate is adjusted between 6.5-7.0 with
any of mineral or organic acids, preferably phosphoric acid, and
beta cyclodextrin is added in the amount 1-5%, preferably 2.0-2.5%,
heated to 50-55.degree.C for about 1-2 hours with agitation. Then,
the solution is cooled to the 10-12.degree.C for about 1 hours. The
formed precipitate is removed by filtration.
[0040] The almost clear solution is mixed with bentonite for water
based systems (Sigma-Aldrich) in the amount 1-5 grams per liter,
preferably about 2-3 grams, and mixed at 40-45.degree.C for about
one hour. The remaining clear solution is drawn off, filtered, and
thickened at 50-55.degree.C, in vacuum to a syrup state.
[0041] The HPLC analysis data of the obtained product, carried out
at Agilent Technologies 1100 Series (USA) equipped with
Zorbax-NH.sub.2 column using acetonitrile-water gradient from
80:20, v/v (2 minutes) to 50:50, v/v during 70 minutes, or
acetonitrile-water=70/30, v/v without any gradient and UV detector
at 210 nm is presented in the FIG. 1.
[0042] The obtained syrup is mixed with 96.2% of ethanol and
maintain at 45-50.degree.C for 30 minutes with agitation. The
proportion of syrup and ethanol is between 1:2-1:7, w/v, preferably
1:5. During this time the precipitate is formed, which is filtered
and dried. According to the HPLC analysis the powder contents
around 83-84% of rebaudioside A (FIG. 2). For the further
purification the powder is mixed with five volumes of 95% of
ethanol, and treated as in the case of syrup. The rebaudioside A
with 98.5% of purity is obtained (FIG. 3).
[0043] The liquid filtrate is used for the next step to recover the
stevioside (FIG. 4).
[0044] The filtrate is mixed with bentonite for organic systems
(Sigma-Aldrich) in the amount 1-5 grams per liter, preferably about
2-3 grams, and mixed at 40-45.degree.C for about one hour. The
suspension is filtered, mixed with equal volume of distilled water,
and the ethanol is removed by vacuum evaporation. Deionization of
the resulted solution is developed by conventional methods using
ion exchange resins, such as Dowex 50WX4-200 (H type) and Amberlite
IRA96 (OH type), concentrated and dried. The resulted powdered
stevioside have about 93% of purity (FIG. 5). For the further
purification the powder is mixed with two volumes of 90% of
ethanol, and at 10-12.degree.C. maintained for about 30 minutes
with slow agitation. The precipitate is separated by filtration and
dried under vacuum. The stevioside with about 98.0-98.5% of purity
is obtained (FIG. 6).
[0045] For the production of the purified stevia extract only
without separation of individual compounds the solution after
removing the leaves and treatment by calcium hydroxide,
cyclodextrin, and bentonite is deionized, concentrated and
dried.
[0046] The enzymatic transglycosylation of sweeteners obtained is
developed with CGTases produced by cultures Thermoactinomyces
vulgaris INMIA (Institute of microbiology of the National Academy
of Sciences of Armenia)-Tac-3554 and Bacillus halophilus BIO-12H.
CGTase producers have been identified among thermoactinomycets and
halophilic bacilli for the first time.
[0047] The colonies of T. vulgaris fast growing, flat at
50.degree.C, with moderate covering of white mycelium and a
feathery margin on used nutrient medium. The colony reverse is
white. No soluble pigments are produced.
[0048] Substrate mycelium well-developed, branched, septate,
0.6-0.75 .mu.m in diameter. Aerial mycelium 0.7-0.9 .mu.m diameter.
Spores formed singly on aerial and substrate hyphae, spheroidal,
0.53-1.0 .mu.m in diameter.
[0049] The colonies of B. halophilus on the nutrient agar medium at
37.degree.C. are round with entire margins, cream-colored, flat,
not-brilliant.
[0050] The morphological and physiological characteristics of the
strains are presented in the TABLE 1, FIG. 10 and FIG. 11.
TABLE-US-00001 TABLE 1 Characteristics Properties T. vulgaris B.
halophilus Cells size - Rod-shaped, 0.4-0.83 .times. 2.0-4.3 .mu.m,
gram-positive, motile Spore size 0.53-1.0 .mu.m 0.7-0.9 .mu.m
Spores form S S Spore position - T Sporangium swollen - + Catalase
+ + Formation of + + dihydroxiacetone V-P reaction - + Reaction on
lecithinase ND + Nitrate reduction + - Hydrolysis of starch + +
Hydrolysis of hippurate - - Utilization of citrate + - Hydrolysis
of casein + - Hydrolysis of gelatin + - Acid from +/-gas +/-gas
arabinose, xylose, mannitol, glucose, galactose, lactose,
cyclodextrins, maltotriose, mannose, salicin, fructose Growth at pH
5.7 + + Growth at 65. degree. C. + - Growth at 50. degree. C. + +
Anaerobic growth - + Deamination of Phe - - Growth in presence of
NaCl 5% - + 7% - + 10% - + more than 15% - + Formation of urease -
- Formation of indole - - Formation of hydrogen - - sulphide
Degradation of tyrosine + -
[0051] The cultivation of T. vulgaris usable in the present
invention is conducted under aerobic conditions at a temperature
of, usually, 45-60.degree.C., preferably, 49-55.degree.C.; and a pH
of 5-8, preferably, 6.8-7.2. The cultivation time is 20-22 hours.
The aeration rate is adjusted in the range of 0.5:1.0 v/v per one
minute, preferably, 1:1.
[0052] The cultivation of B. halophilus is conducted under aerobic
conditions at a temperature of, usually, 27-40.degree.C.,
preferably, 30-37.degree.C.; and a pH of 5-8, preferably, 6.8-7.2.
The cultivation time is 24-36 hours. The aeration rate is adjusted
in the range of 0.5:1.0 v/v per one minute, preferably, 1:1.
[0053] The cultivation is carried out in a batch-wise or a
continuous manner.
[0054] Any synthetic and natural nutrient culture media can be used
for the cultivation of the microorganisms. Any carbon-containing
substances can be used in the invention as carbon sources. For
instance, saccharides such as sucrose, maltose, dextrin, glucose,
lactose, galactose, cyclodextrins, and starch, as well as
saccharide-containing products such as molasses and yeast extracts,
can be used as the carbon sources. The concentrations of these
carbon sources in nutrient culture media are selectively chosen
depending on their types. However, the best results for both of
microorganisms are obtained in the case of 0.7-2.0% of starch,
preferably, 0.9-1.2%. The nitrogen sources usable in the present
invention are, for example, inorganic nitrogen-containing compounds
such as ammonium salts; and organic nitrogen-containing compounds
such as urea, corn steep liquor, casein, peptone, yeast extract,
and beef extract. The inorganic ingredients usable in the present
invention are, for example, calcium salts, magnesium salts,
potassium salts, sodium salts, phosphates and others.
[0055] Since the CGTase activity is found in the cell-free nutrient
media, the media can be collected and used as a crude enzyme.
Conventional liquid-solid separation methods can be used to remove
cells. For example, methods to directly centrifuge the culture and
those to filtrate with pre-coat filters or to separate cells by
membrane filtration using plain filters or hollow-fibers. The
resulting cell-free culture broth can be used intact as a crude
enzyme, and preferably, used after concentration. The concentration
methods usable in the present invention are, for example, salting
out using ammonium sulfate, sedimentation using acetone and/or
alcohol, and concentration using ultrafiltration membranes such as
plain filters and hollow-fibers.
[0056] Crude enzymes can be immobilized by conventional methods
such as adsorption, covalent biding, and entrapping.
[0057] Crude enzymes can be used intact and after purification. For
example, cell-free culture broth is concentrated using
ultrafiltration membranes, and purified successively on beta
cyclodextrin polymers to obtain an enzyme preparation exhibiting an
electrophoretically single protein band.
[0058] Some properties of the CGTases are presented in TABLE 2.
TABLE-US-00002 TABLE 2 Mol. Thermal Microbial weight, Optimum pH-
stability, Cyclodextrin strains kDa pH stability degree. C.
produced B. halophilus 70 6.5-7.0 6.0-9.0 50 .beta. >>
.gamma. T. vulgaris 64 6.0-7.0 5.5-8.5 70 .beta. > .alpha. >
.gamma.
[0059] The amino acid composition of the enzymes is presented in
the TABLE 3. TABLE-US-00003 TABLE 3 Number of residues, % Amino
acides T. vulgaris B. halophilus Asp + Asn 18.3 17.4 Thr 8.7 9.1
Ser 6.3 7.5 Glu + Gln 8.2 5.5 Pro 5.0 4.5 Gly 9.5 9.8 Ala 8.0 9.4
Cys/2 0.2 0.4 Val 6.2 6.6 Met 1.6 3.3 Ile 4.7 5.0 Leu 5.5 5.3 Tyr
4.4 4.1 Phe 4.8 4.8 Lys 3.8 3.2 Trp ND ND His 2.6 1.8 Arg 2.2
2.3
[0060] The activity of CGTases according to the present invention
is assayed as follows: The mixture of 10 .mu.l enzyme and 0.2%
amylose solution in 0.2M buffer is incubated at 50.degree. C. for
10 min. The reaction is stopped by adding 1 ml 0.5M acetic acid and
0.5 ml 0.02% I.sub.2/0.2% KI solution. The mixture volume is
brought up to 10 ml with distilled water, and extinction is
determined under 700 nm. The enzyme activity unit is accepted as
the enzyme quantity that has reduced the intensity of blue colour
by 10% for 1 min.
[0061] The present CGTases acts to the mixture of starch and
stevioside, rebaudioside A or the purified Stevia extract to
produce alpha-glucosylated stevioside and alpha-glucosylated
rebaudioside A.
[0062] Starch of various origins, for example, from wheat, corn,
potato, tapioca, and sago can be used.
[0063] The dextrose equivalent of the partially hydrolyzed starch
can be in the range of 5-50, preferably 6-10.
[0064] The amount of enzyme to be used for liquefaction of starch
is in the limits of 1-5 units/gram of starch, preferably 2-3 units.
In the stage of transglycosylation the quantity of enzyme is 7-15
units/gram of starch, preferably 8-11 units. However, the larger
the amount of enzyme, the higher the yield of transglycosylated
products and the shorter the duration of enzymatic reaction.
[0065] The process temperature is 45-70.degree.C, preferably
55-60.degree.C. The reaction rate is increased with increasing
reaction temperature. Low temperatures are not favorable.
[0066] The following examples illustrate preferred embodiments of
the invention.
[0067] Experiment 1
[0068] Extraction of Sweet Glycosydes
[0069] The leaves of Stevia rebaudiana are dried at 55.degree.C.
for three hour in vacuum oven and powdered (30 mesh). One kg of the
obtained material was mixed with 10 liters of water (pH 6.5) and
heated to 55.degree.C. with uninterruptedly agitation. 20 grams of
PECTINEX Ultra-SP-L is added to the suspension and extraction is
carried out at 60.degree.C. for 5 hours. The plant material is
separated from the solution by filtration and the pH of the
filtrate is adjusted to 10 with calcium hydroxide and heated to
55.degree.C. for 1.0 hours, cooled to ambient temperature with slow
agitation, and the resulted residue is removed by filtration.
[0070] The pH of resulted filtrate is adjusted to about 7.0 with
phosphoric acid, and 220 grams of beta cyclodextrin is added. The
solution is maintained at 55.degree.C for 1.5 hours with agitation,
then cooled to about 10.degree.C for 1 hour, and the formed
precipitate is removed by filtration.
[0071] The filtrate is collected, mixed with 200 grams of bentonite
for water based systems (Sigma-Aldrich), and maintained at
40.degree.C for one hour. The precipitate is removed by filtration
and the resulted clear solution is concentrated at 50-55.degree.C.,
in vacuum to a syrup state.
[0072] In the case for producing the purified extract only without
the separation of stevioside and rebaudioside, the solution after
bentonite removal is deionized by conventional ion exchange
chromatography on Dowex 50WX4-200 (H.sup.+) and Amberlite IRA96
(OH.sup.-), concentrated and dried. The yield was 116 grams with
content of sweet glycosides to about 95%. The mixture contains
Dulcoside, 3.2%; Stevioside, 60.4%; Rebaudioside C, 5.7%; and
Rebaudioside A, 25.7%.
[0073] Experiment 2
[0074] Separation and Purification of Rebaudioside A
[0075] Fifty grams (on the base of dry material) of the syrup
obtained by the process of EXPERIMENT 1 is mixed with 0.25 liters
of 96.2% of ethanol and heated to about 50.degree.C for 30 minutes
with slow agitation. The mixture is cooled to the ambient
temperature; the resulted precipitate is separated by filtration
and dried. The powder weighed 14.2 grams and contained Rebaudioside
A, 84.4%; Stevioside, 11.2%; and Rebaudioside C, 4.4%. For the
further purification the powder is mixed with five volumes of 95%
of ethanol, and treated as in the case of initial syrup. 10.1 grams
of rebaudioside A with more than 98% of purity is obtained.
[0076] Experiment 3
[0077] Separation and Purification of Stevioside
[0078] The filtrate obtained in the EXPERIMENT 2 containes
Stevioside, 87.7%; rebaudioside A, 0.65%; rebaudioside C, 6.8%; and
Dulcoside, 4.8%. of is mixed with 2.0 w/v % of bentonite for
organic systems (Sigma-Aldrich) and 2% (w/v) of beta cyclodextrin
and heated at 40.degree.C for about one hour. The suspension is
filtered, mixed with equal volume of distilled water, and the
ethanol is removed by vacuum evaporation. Deionization of the
resulted solution is developed by conventional methods using ion
exchange resins, such as Dowex 50WX4-200 (H type) and Amberlite
IRA96 (OH type), concentrated and dried. The resulted powder
weighed 28.7 grams and contains about 93% of stevioside. For the
further purification the powder is mixed with two parts of 90% of
ethanol, and at 10-12.degree.C. maintained for about 30 minutes
with slow agitation. The precipitate is separated by filtration and
dried under vacuum. The product weighed 26.7 grams and contains
98.3% of stevioside.
[0079] Experiment 4
[0080] Preparation of CGTase of Thermoactonomyces vulgaris
[0081] A strain of Thermoactonomyces vulgaris INMIA-Tac-3554 was
inoculated on 10 liters of a sterilized culture medium (pH 7.0-7.2)
containing 1.0% (w/v) soluble starch; 0.5% (w/v) corn steep liquor;
0.5% (w/v) sodium chloride; 0.5% (w/v) peptone; and 0.5% (w/v)
calcium carbonate, and the mixture was incubated at a temperature
of 50.degree. C. for 20 hours with aeration and stirring. The
resultant culture broth was centrifuged and the supernatant was
concentrated up to five times on ultrafiltration membranes. The
concentrated solution is diluted with three volumes of distilled
water and again concentrated to the initial concentrate volume. The
process is repeated for three times. A crude enzyme preparation
with an activity of about 60,000 units was obtained.
[0082] Experiment 5
[0083] Preparation of CGTase of Bacillus halophilus
[0084] A strain of Bacillus halophilus BIO-12H was inoculated on 10
liters of a sterilized culture medium (pH 7.0-7.2) containing 1.0%
(w/v) soluble starch; 0.5% (w/v) corn steep liquor; 1.0% (w/v)
peptone; 10.0% (w/v) sodium chloride; 2.0% (w/v) potassium
chloride; 2.0% (w/v) magnesium sulphate heptahydrate; 1.0% (w/v)
manganess sulphate; and 0.5% (w/v) calcium carbonate, and the
mixture was incubated at a temperature of 37.degree. C. for 24
hours with aeration and stirring. The resultant culture broth was
centrifuged and the supernatant was concentrated up to five times
on ultrafiltration membranes. The concentrated solution is diluted
with three volumes of distilled water and again concentrated to the
initial concentrate volume. The process is repeated for three
times. A crude enzyme preparation with an activity of about 40,000
units was obtained.
[0085] Experiment 6
[0086] Purification of Enzymes
[0087] The purification procedure was carried out as follows.
Concentrated culture broth (100 ml; 10-11 mg protein per one ml)
was mixed with 5 g beta cyclodextrin polymer at 4-5.degree.C. for
16-18 hours. The mixture was centrifuged, and the residue carefully
washed in sequence with distilled water, 1M NaCl, and distilled
water. The adsorbed enzyme was eluted with 5 mM alpha cyclodextrin
in aqueous 0.5 M NaCl at ambient temperature for 3 hours. The
eluate was dialyzed against deionized water for 12-14 hours and
lyophilized.
[0088] At this stage the enzymes from halophilic strain and
thermoactinomycete could be purified 53.2- and 57.4-fold,
respectively. The degree of the enzyme purification was
considerably higher than in the case of adsorption on starch. The
method can be used under continuous-flow conditions.
[0089] The enzyme solution (10-11 mg lyophilized powder per one ml)
was passed through the column (1.6.times.20 cm) packed and
equilibrated with DEAE-.beta.-CD-cellulose copolymer and 0.01 M
phosphate buffer (pH 7.0), respectively. The column was washed with
0.5 M NaCl, and the proteins were eluted using a linear gradient of
alpha cyclodextrin (from 0 to 10 mM) in 0.01 M phosphate buffer (pH
7.0) containing 0.2 M NaCl. The collected active fractions were
dialyzed against 0.01 M phosphate buffer (pH 7.0) containing 1 mM
calcium chloride, and the dialyzed fraction was lyophilized.
[0090] The yield of enzyme preparations of halophilic strain and
thermoactinomycete was about 17% and 15% with the specific activity
76.3 units/mg protein and 95.4 units/mg protein respectively. The
protein content was assayed by the Lowry method using serum albumin
as a standard protein.
[0091] The purity of the enzymes was assayed by gel electrophoresis
in 7.5% w/v % polyacrylamide gel, resulting in a single protein
band.
[0092] The polymer of beta cyclodextrin have been prepared as
follows: 55 g of beta cyclodextrin is dissolved in 50 ml of 50% of
NaOH solution with the content of 50 mg of NaBH.sub.4 at
12-15.degree.C. Then 34 ml of epichlorohydrin is added and the
mixture is vigorously stirred at 50.degree.C. for 30-45 min. The
resulting gel is suspended in 1000 ml of water with the pH 2-2.5
and boiled with vigorously mixing for 5 min. After that the polymer
is separated by filtration, washed with distilled water to pH
6.5-7.0 and dried.
[0093] The DEAE polymer of beta cyclodextrin have been prepared as
follows: Ten grams of beta cyclodextrin polymer is dried at
80.degree.C. for 14-16 hours and then suspended in the mixture of
50 ml dimethylsulfoxide and 24 g of fine powdered NaOH. After
stirring at 60.degree.C. for 1-1.5 hours, 30 g of
2-chlorotriethylamine hydrochloride is added and stirred at room
temperature for 30 min. The precipitate is separated by filtration,
washed with distilled water until neutral reaction and then
dried.
[0094] Experiment 7
[0095] Enzymatic Treatment of Stevioside, Rebaudioside A and
Purified Stevia Extract
[0096] 100 grams of tapioca starch was suspended in 600 ml of
distilled water (pH 6.5-7.0), 200 units of crude CGTase obtained in
Experiment 4 or 5 was added, and the liquefaction of starch was
carried out at 75-80.degree.C. for about one hour to dextrose
equivalent about 10. After cooling to 50-60.degree.C., rebaudioside
A (obtained in Experiment 2) or stevioside (obtained in Experiment
3) or purified Stevia mixture (obtained in Experiment 1) in the
amounts of 100 grams was added and mixed until homogeneous solution
is obtained. Then, 800 units of crude CGTase were added to the
solution, allowed incubation at a temperature of 55.degree. C. for
48 hours, and heated at 100.degree.C. for 10 minutes to inactivate
the enzyme. The resulted reaction mixture was decolorized with 1%
(w/v) activated carbon, the solution was concentrated at a
temperature of 65-70.degree.C. under reduced pressure, and
dried.
REFERENCES
[0097] Chang, S. S. and Cook, J. M. 1983. Stability studies of
stevioside and rebaudioside A in carbonated beverages. J. Agric.
Food Chem. 31: 409-412. [0098] Dobberstein, R. H., and Ahmad, M. S.
1982. Extraction, separation and recovery of diterpene glycosides
from Stevia rebaudiana plants. U.S. Pat. No. 4,361,697. [0099]
DuBois, G. E. and Stephenson, R. A. 1984. Diterpenoid sweeteners.
Synthesis and sensory evaluation of stevioside analogues with
improved organoleptic properties. J. Med. Chem 28:93-98. [0100]
Giovanetto, R. H. 1990. Method for the recovery of steviosides from
plant raw material. U.S. Pat. No. 4,892,938. [0101] Kitahata, S.,
Ishikawa, S., Miyata, T. and Tanaka, O. 1989. Production of
rubusoside derivatives by transglycosylation of various
beta-galactosidase. Agricultural and Biological Chemistry. 53:
2923-2928. [0102] Kutowy, O., Zhang, Q. S., and Kumar, A. 1999.
Extraction of sweet compounds from Stevia rebaudiana Bertoin. U.S.
Pat. No. 5,972,120. [0103] Lobov, S. V., Jasai, R., Ohtani, K.,
Tanaka, O. and Yamasaki, K. 1991. Enzymatic production of sweet
stevioside derivatives: transglycosylation by glucosidases.
Agricultural and Biological Chemistry. 55: 2959-2965. [0104]
Miyake, T. 1980. Process for producing a sweetener. U.S. Pat. No.
4,219,571. [0105] Morita, T., Fujita, I., and Iwamura, J. 1979.
Sweetening compound, method of recovery, and use thereof. U.S. Pat.
No. 4,082,858. [0106] Tanaka, O. 1987. Improvement of taste of
natural sweetners. Pure Appl. Chem 69:675-683. [0107] Payzant, J.
D., Laidler, J. K., and Ippolito, R. M. 1999. Method of extracting
selected sweet glycosides from the Stevia rebaudiana plant. U.S.
Pat. No. 5,962,678. [0108] Phillips, K. C. 1989. Stevia: steps in
developing a new sweetener. Pages 1-43 in T. H. Grenby ed.
Developments in sweeteners, Volume 3. Elsivier Applied Science,
London. [0109] Schiffman, S. S., Booth, B. J., Carr, B. T., Losee,
M. L., Sattely-Miller, E., and Graham, B. G. 1995. Investigation of
synergism in binary mixtures of sweeteners. Brain Res. Bull. 38:
105-120. [0110] Yamamoto, K., Yoshikawa, K. and Okada, S. 1994.
Effective production of glucosyl-stevioside by
alpha-1,6-transglucosylation of dextran dextranase. Bioscience,
Biotechnology, and Biochemistry. 58: 1657-1661.
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